Products for inhibiting expression of human MDM2

ABSTRACT

A human gene has been discovered which is genetically altered in human tumor cells. The genetic alteration is gene amplification and leads to a corresponding increase in gene products. Detecting that the gene, designated hMDM2, has become amplified or detecting increased expression of gene products is diagnostic of tumorigenesis. Human MDM2 protein binds to human p53 and allows the cell to escape from p53-regulated growth.

This application is a continuation application U.S. Ser. No. 08/390,474,filed Feb. 17, 1995, which is a divisional of U.S. Ser. No. 08/044,619,filed Apr. 7, 1993, now U.S. Pat. No. 5,420,263 which is acontinuation-in-part of U.S. Ser. No. 07/903,103, filed Jun. 23, 1992,now U.S. Pat. No. 5,411,860 which is a continuation-in-part of U.S. Ser.No. 07/867,840, filed Apr. 7, 1992, now abandoned.

This invention was made with support from the U.S. Government, includingNIH grants CA-57345, CA-43460, CA-02243 and CA-35494. Accordingly, theGovernment retains certain rights in the invention.

FIELD OF THE INVENTION

The invention relates to the area of cancer diagnostics andtherapeutics. More particularly, the invention relates to the detectionof a gene which is amplified in certain human tumors.

BACKGROUND OF THE INVENTION

According to the Knudson model for tumorigenesis (Cancer Research, 1985,vol. 45, p. 1482), there are tumor suppressor genes in all normal cellswhich, when they become non-functional due to mutation, cause neoplasticdevelopment. Evidence for this model has been found in cases ofretinoblastoma and colorectal tumors. The implicated suppressor genes inthese tumors, RB and p53 respectively, were found to be deleted oraltered in many of the tumors studied.

The p53 gene product, therefore, appears to be a member of a group ofproteins which regulate normal cellular proliferation and suppression ofcellular transformation. Mutations in the p53 gene have been linked totumorigenesis, suggesting that alterations in p53 protein function areinvolved in cellular transformation. The inactivation of the p53 genehas been implicated in the genesis or progression of a wide variety ofcarcinomas (Nigro et al., 1989, Nature 342:705-708), including humancolorectal carcinoma (Baker et al., 1989, Science 244:217-221), humanlung cancer (Takahashi et al., 1989, Science 246:491494; Iggo et al.,1990, Lancet 335:675-679), chronic myelogenous leukemia (Kelman et al,1989, Proc. Natl. Acad. Sci. USA 86:6783-6787) and osteogenic sarcomas(Masuda et al., 1987, Proc. Natl. Acad. Sci. USA 84:7716-7719).

While there exists an enormous body of evidence linking p53 genemutations to human tumorigenesis (Hollstein et al., 1991, Science253:49-53) little is known about cellular regulators and mediators ofp53 function.

Hinds et al. (Cell Growth & Differentiation, 1:571-580, 1990), foundthat p53 cDNA clones, containing a point mutation at amino acid residue143, 175, 273 or 281, cooperated with the activated ras oncogene totransform primary rat embryo fibroblasts in culture. These mutant p53genes are representative of the majority of mutations found in humancancer. Hollstein et al., 1991, Science 253:49-53. The transformedfibroblasts were found to produce elevated levels of human p53 proteinhaving extended half-lives (1.5 to 7 hours) as compared to the normal(wild-type) p53 protein (20 to 30 minutes).

Mutant p53 proteins with mutations at residue 143 or 175 form anoligomeric protein complex with the cellular heat shock protein hsc70.While residue 273 or 281 mutants do not detectably bind hsc70, and arepoorer at producing transformed foci than the 175 mutant, complexformation between mutant p53 and hsc70 is not required for p53-mediatedtransformation. Complex formation does, however, appear to facilitatethis function. All cell lines transformed with the mutant p53 genes aretumorigenic in a thymic (nude) mice. In contrast, the wild-type humanp53 gene does not possess transforming activity in cooperation with ras.Tuck and Crawford, 1989, Oncogene Res. 4:81-96.

Hinds et al., supra also expressed human p53 protein in transformed ratcells. When the expressed human p53 was immunoprecipitated with two p53specific antibodies directed against distinct epitopes of p53, anunidentified M, 90,000 protein was coimmunoprecipitated. This suggestedthat the rat M, 90,000 protein is in a complex with the human p53protein in the transformed rat cell line.

As mentioned above, levels of p53 protein are often higher intransformed cells than normal cells. This is due to mutations whichincrease its metabolic stability (Oven et al., 1981, Mol. Cell. Biol.1:101-110; Reich et al. (1983), Mol. Cell. Biol. 3:2143-2150). Thestabilization of p53 has been associated with complex formation betweenp53 and viral or cellular proteins. (Linzer and Levine, 1979, Cell17:43-52; Crawford et al., 1981, Proc. Natl. Acad. Sci. USA 78:41-45;Dippold et al., 1981, Proc. Natl. Acad. Sci. USA 78:1695-1699; Lane andCrawford, 1979, Nature (Lond.) 278:261-263; Hinds et al., 1987, Mol.Cell. Biol. 7:2863-2869; Finlay et al., 1988, Mol. Cell. Biol.8:531-539; Sarnow et al., 1982, Cell. 28:387-394; Gronostajski et al.,1984, Mol. Cell. Biol. 4:442-448; Pinhasi-Kimhi et al., 1986, Nature(Lond.) 320.182-185; Ruscetti and Scolnick, 1983, J. Virol.46:1022-1026; Pinhasi and Oren, 1984, Mol. Cell. Biol. 4:2180-2186; andSturzbecher et al., 1987, Oncogene 1:201-211.) For example, p53 proteinhas been observed to form oligomeric protein complexes with the SV40large T antigen, the adenovirus type 5 E1B-M_(r) 55,000 protein, and thehuman papilloma virus type 16 or 18 E6 product. Linzer and Levine, 1979,Cell 17:43-52; Lane and Crawford, 1979, Nature, 278:261-263; Sarnow etal., 1982, Cell 28:387-394; Werness et al., 1990, Science, 248:76-79.Similarly, complexes have been observed of p₁₀₅ ^(RB) (the product ofthe retinoblastoma susceptibility gene) with T antigen (DeCaprio et al.,1988, Cell 54:275-283), the adenovirus EIA protein (Whyte et al., 1988,Nature 334:124-129) and the E7 protein of human papilloma virus 16 or 18(Munger et al., 1989, EMBO J. 8:4099-4105). It has been suggested thatinteractions between these viral proteins and p105^(RB) inactivate agrowth-suppressive function of p105^(RB), mimicking deletions andmutations commonly found in the RB gene in tumor cells. In a similarfashion, oligomeric protein complex formation between these viralproteins and p53 may eliminate or alter the function of p53. Finlay etal., 1989, Cell 57:1083-1093.

Fakharzadeh et al. (EMBO J. 10:1565-1569, 1991) analyzed amplified DNAsequences present in a tumorigenic mouse cell line (i.e., 3T3DM, aspontaneously transformed derivative of mouse Balb/c cells). Studieswere conducted to determine whether any of the amplified genes inducedtumorigenicity following introduction of the amplified genes into anontransformed recipient cell (e.g., mouse NIH3T3 or Rat2 cells). Theresulting cell lines were tested for tumorigenicity in nude mice. Agene, designated MDM2, which is amplified more than 50 fold in 3T3DMcells, induced tumorigenicity when overexpressed in NIH3T3 and Rat 2cells. From the nucleotide and predicted amino acid sequence of mouseMDM2 (mMDM2), Fakharzadeh speculated that this gene encodes a potentialDNA binding protein that functions in the modulation of expression ofother genes and, when present in excess, interferes with normalconstraints on cell growth.

SUMMARY OF THE INVENTION

It is an object of the invention to provide a method for diagnosing aneoplastic tissue, such as sarcoma, in a human.

It is another object of the invention to provide a CDNA moleculeencoding the sequence of human MDM2.

Yet another object of the invention is to provide a preparation of humanMDM2 protein which is substantially free of other human cellularproteins.

Still another object of the invention is to provide DNA probes capableof hybridizing with human MDM2 genes or MRNA molecules.

Another object of the invention is to provide antibodies immunoreactivewith human MDM2 protein.

Still another object of the invention is to provide kits for detectingamplification or elevated expression of human MDM2.

Yet another object of the invention is to provide methods foridentifying compounds which interfere with the binding of human MDM2 tohuman p53.

A further object of the invention is to provide a method of treating aneoplastic human cell.

Yet another object of the invention is to provide methods for inhibitingthe growth of tumor cells which contain a human MDM2 gene amplification.

Still another object of the invention is to provide polypeptides whichinterfere with the binding of human MDM2 to human p53.

It has now been discovered that hMDM2, a heretofore unknown human gene,plays a role in human cancer. The hMDM2 gene has been cloned and therecombinant derived hMDM2 protein shown to bind to human p53 in vitro.hMDM2 has been found to be amplified in some neoplastic cells and theexpression of hMDM2-encoded products has been found to becorrespondingly elevated in tumors with amplification of this gene. Theelevated levels of MDM2 appear to sequester p53 and allow the cell toescape from p53-regulated growth.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C shows the CDNA sequence of human MDM2. In this figure, humanand mouse nucleotide and amino acid sequences are compared, the mousesequence being shown only where it differs from the corresponding humansequence.

FIG. 2 shows that hMDM2 binds to p53.

FIGS. 3A and 3B illustrate the amplification of the hMDM2 gene insarcomas.

FIGS. 4A-C illustrates hMDM2 expression.

FIG. 5A shows the inhibition of p53-mediated transactivation by MDM2.Yeast were stably transfected with expression plasmids encoding p53,lex-VP16, MDM2 or the appropriate vector-only controls, as indicated.p53-responsive (bars a-c) or lexA-responsive (bars d-f) β-galactosidasereporter plasmids were used to assess the response. Inset: Western blotanalysis demonstrating MDM2 (90 kD) and p53 (53 kD) expression inrepresentative yeast strains. The strain indicated by a plus wastransfected with expression vector encoding full length MDM2 and p53,while the strain indicated by a minus was transfected only with the p53expression vector.

FIG. 6 shows the determination of MDM2 and p53 domains of interaction.FIG. 6A and FIG. 6B. Random fragments of MDM2 were fused to sequencesencoding the lexA DNA binding domain and the resultant clonestransfected into yeast carrying pRS314SN (p53 expression vector) andpJK103 (lexA-responsive β-galactosidase reporter). Yeast clonesexpressing β-galactosidase were identified by their blue color, and theMDM2 sequences in the lexA fusion vector were determined.β-galactosidase activity was observed independent of p53 expression inA, but was dependent on p53 expression in B. The bottom 6 clones in Bwere generated by genetic engineering. FIG. 6C. Random fragments of p53were fused to the sequence encoding the B42 acidic activation domain anda hemagglutinin epitope tag; the resultant clones were transfected intoyeast carrying lexA-MDM2 (lexA DNA binding domain fused to full lengthMDM2) and pJK103. Yeast clones were identified as above, and all werefound to be MDM2-dependent. The bottom three clones were generated bygenetic engineering.

FIGS. 7A, 7B, 7C, and 7D show protein expression from the yeast strainsdescribed in FIG. 6. Western blot analysis was performed as described(Oliner, J. D., et al., Nature 358:80-83 (1992)), using 20 μg of proteinper lane. The MDM2 and p53 codons contained in the fusion vectors areshown at the top of A and B, respectively. FIG. 7A. Upper panel probedwith p53 Ab2 detecting p53; lower panel probed with anti-lexA polyclonalantibodies (lex Ab) detecting MDM2 fusion proteins of 30-50 kD. FIG. 7B.Upper panel probed with Lex Ab detecting the lexA-full length MDM2fusion protein of 112 kD; lower panel probed with HA Ab (a monoclonalantibody directed against the hemagglutinin epitope tag, BerkeleyAntibody) detecting p53 fusion proteins of approximately 25-30 kD.

FIG. 8A shows the inhibition of the p53 activation domain by MDM2. Yeastwere transfected with expression vectors encoding a lexA-p53 (p53 codons1-73) fusion (bars a and b) or lexA alone (bar c). Strain b alsoexpressed full length MDM2, and all strains contained thelexa-responsive β-galactosidase reporter plasmid. Inset: Upper panelprobed with MDM2 polyclonal antibodies detecting full length MDM2 (90kD); lower panel probed with lex Ab detecting the lex-p53 fusion proteinof 40 kD.

DETAILED DESCRIPTION OF THE INVENTION

It is a discovery of the present invention that a gene exists which isamplified in some human tumors. The amplification of this gene,designated MDM2, is diagnostic of neoplasia or the potential therefor.Detecting the elevated expression of human MDM2-encoded products is alsodiagnostic of neoplasia or the potential for neoplastic transformation.Over a third of the sarcomas surveyed, including the most common boneand soft tissue forms, were found to have amplified hMDM2 sequences.Expression of hMDM2 was found to be correspondingly elevated in tumorswith the gene amplification.

Other genetic alterations leading to elevated hMDM2 expression may beinvolved in tumorigenesis also, such as mutations in regulatory regionsof the gene. Elevated expression of hMDM2 may also be involved in tumorsother than sarcomas including but not limited to those in which p53inactivation has been implicated. These include colorectal carcinoma,lung cancer and chronic myelogenous leukemia.

According to one embodiment of the invention, a method of diagnosing aneoplastic tissue in a human is provided. Tissue or body fluid isisolated from a human, and the copy number of human MDM2 genes isdetermined. Alternatively, expression levels of human MDM2 gene productscan be determined. These include protein and mRNA.

Body fluids which may be tested include urine, serum, blood, feces,saliva, and the like. Tissues suspected of being neoplastic aredesirably separated from normal appearing tissue for analysis. This canbe done by paraffin or cryostat sectioning or flow cytometry, as isknown in the art. Failure to separate neoplastic from non-neoplasticcells can confound the analysis. Adjacent non-neoplastic tissue or anynormal tissue can be used to determine a base-line level of expressionor copy number, against which the amount of hMDM2 gene or gene productscan be compared.

The human MDM2 gene is considered to be amplified if the cell containsmore than the normal copy number (2) of this gene per genome. Thevarious techniques for detecting gene amplification are well known inthe art. Gene amplification can be determined, for example, by Southernblot analysis, as described in Example 4, wherein cellular DNA from ahuman tissue is digested, separated, and transferred to a filter whereit is hybridized with a probe containing complementary nucleic acids.Alternatively, quantitative polymerase chain reaction (PCR) employingprimers can be used to determine gene amplification. Appropriate primerswill bind to sequences that bracket human MDM2 coding sequences. Othertechniques for determining gene copy number as are known in the art canbe used without limitation.

The gene product which is measured may be either mRNA or protein. Theterm elevated expression means an increase in mRNA production or proteinproduction over that which is normally produced by non-cancerous cells.Although amplification has been observed in human sarcomas, othergenetic alterations leading to elevated expression of MDM2 may bepresent in these or other tumors. Other tumors include those of lung,breast, brain, colorectal, bladder, prostate, liver, skin, and stomach.These, too, are contemplated by the present invention. Non-cancerouscells for use in determining base-line expression levels can be obtainedfrom cells surrounding a tumor, from other humans or from human celllines. Any increase can have diagnostic value, but generally the mRNA orprotein expression will be elevated at least about 3-fold, 5-fold, andin some cases up to about 100-fold over that found in non-cancerouscells. The particular technique employed for detecting mRNA or proteinis not critical to the practice of the invention. Increased productionof MRNA or protein may be detected, for example, using the techniques ofNorthern blot analysis or Western blot analysis, respectively, asdescribed in Example 4 or other known techniques such as ELISA,immunoprecipitation, RIA and the like. These techniques are also wellknown to the skilled artisan.

According to another embodiment of the invention, nucleic acid probes orprimers for the determining of human MDM2 gene amplification or elevatedexpression of mRNA are provided. The probe may comprise ribo- ordeoxyribonucleic acids and may contain the entire human MDM2 codingsequence, a sequence complementary thereto, or fragments thereof. Aprobe may contain, for example, nucleotides 1-949, or 1-2372 as shown inFIG. 1. Generally, probes or primers will contain at least about 14contiguous nucleotides of the human sequence but may desirably containabout 40, 50 or 100 nucleotides. Probes are typically labelled with afluorescent tag, a radioisotope, or the like to render them easilydetectable. Preferably the probes will hybridize under stringenthybridization conditions. Under such conditions they will not hybridizeto mouse MDM2. The probes of the invention are complementary to thehuman MDM2 gene. This means that they share 100% identity with the humansequence.

hMDM2 protein can be produced, according to the invention, substantiallyfree of other human proteins. Provided with the DNA sequence, those ofskill in the art can express the cDNA in a non-human cell. Lysates ofsuch cells provide proteins substantially free of other human proteins.The lysates can be further purified, for example, byimmunoprecipitation, co-precipitation with p53, or by affinitychromatography.

The antibodies of the invention are specifically reactive with hMDM2protein. Preferably, they do not cross-react with MDM2 from otherspecies. They can be polyclonal or monoclonal, and can be raised againstnative hMDM2 or a hMDM2 fusion protein or synthetic peptide. Theantibodies are specifically immunoreactive with hMDM2 epitopes which arenot present on other human proteins. Some antibodies are reactive withepitopes unique to human MDM2 and not present on the mouse homolog. Theantibodies are useful in conventional analyses, such as Western blotanalysis, ELISA, immunohistochemistry, and other immunological assaysfor the detection of proteins. Techniques for raising and purifyingpolyclonal antibodies are well known in the art, as are techniques forpreparing monoclonal antibodies. Antibody binding can be determined bymethods known in the art, such as use of an enzyme-labelled secondaryantibody, staphylococcal protein A, and the like. Certain monoclonalantibodies of the invention, have been deposited at the American TypeCulture Collection, 12301 Parklawn Drive, Rockville, Md. 20852. Theseinclude IF2, and ED9, which have been granted accession Nos. HB 11290,and HB 11291, respectively.

According to another embodiment of the invention, interference with theexpression of MDM2 provides a therapeutic modality. The method can beapplied in vivo, in vitro, or ex vivo. For example, expression may bedown regulated by administering triple-strand forming or antisenseoligonucleotides which bind to the hMDM2 gene or mRNA, respectively andprevent transcription or translation. The oligonucleotides may interactwith unprocessed pre-mRNA or processed mRNA. Small molecules andpeptides which specifically inhibit MDM2 expression can also be used.Similarly, such molecules which inhibit the binding of MDM2 to p53 wouldbe therapeutic by alleviating the sequestration of p53.

Such inhibitory molecules can be identified by screening forinterference of the hMDM2/p53 interaction where one of the bindingpartners is bound to a solid support and the other partner is labeled.Antibodies specific for epitopes on hMD2 or p53 which are involved inthe binding interaction will interfere with such binding. Solid supportswhich may be used include any polymers which are known to bind proteins.The support may be in the form of a filter, column packing matrix,beads, and the like. Labeling of proteins can be accomplished accordingto any technique known in the art. Radiolabels, enzymatic labels, andfluorescent labels can be used advantageously. Alternatively, both hMDM2and p53 may be in solution and bound molecules separated from unboundsubsequently. Any separation technique known in the art may be employed,including immunoprecipitation or immunoaffinity separation with anantibody specific for the unlabeled binding partner.

It has been found that amino acid residues 1341 of p53 (See SEQ IDNO: 1) are necessary for the interaction of MDM-2 and p53. However,additional residues on either the amino or carboxy terminal side of thepeptide appear also to be required. Nine to 13 additional p53 residuesare sufficient to achieve MDM2 binding, although less may be necessary.Since cells which overexpress MDM2 escape from p53-regulated growthcontrol in sarcomas, the use of p53-derived peptides to bind to excessMDM2 leads to reestablishment of p53-regulated growth control.

Suitable p53-derived peptides for administration are those which arecircular, linear, or derivitized to achieve better penetration ofmembranes, for example. Other organic compounds which are modelled toachieve the same three dimensional structure as the peptide of theinvention can also be used.

DNA encoding the MDM2-binding, p53-derived peptide, or multiple copiesthereof, may also be administered to tumor cells as a mode ofadministering the peptide. The DNA will typically be in an expressionconstruct, such as a retrovirus, DNA virus, or plasmid vector, which hasthe DNA elements necessary for expression properly positioned to achieveexpression of the MDM2-binding peptide. The DNA can be administered,inter alia encapsulated in liposomes, or in any other form known to theart to achieve efficient uptake by cells. As in the directadministration of peptide, the goal is to alleviate the sequestration ofp53 by MDM2.

A cDNA molecule containing the coding sequence of hMDM2 can be used toproduce probes and primers. In addition, it can be expressed in culturedcells, such as E. coli, to yield preparations of hMDM2 proteinsubstantially free of other human proteins. The proteins produced can bepurified, for example, with immunoaffinity techniques using theantibodies described above.

Kits are provided which contain the necessary reagents for determininggene copy number, such as probes or primers specific for the hMDM2 gene,as well as written instructions. The instructions can providecalibration curves to compare with the determined values. Kits are alsoprovided to determine elevated expression of MRNA (i.e., containingprobes) or hMDM2 protein (i.e., containing antibodies). Instructionswill allow the tester to determine whether the expression levels areelevated. Reaction vessels and auxiliary reagents such as chromogens,buffers, enzymes, etc. may also be included in the kits.

The human MDM2 gene has now been identified and cloned. Recombinantderived hMDM2 has been shown to bind to human p53. Moreover, it has beenfound that hMDM2 is amplified in some sarcomas. The amplification leadsto a corresponding increase in MDM2 gene products. Such amplification isassociated with the process of tumorigenesis. This discovery allowsspecific assays to be performed to assess the neoplastic or potentialneoplastic status of a particular tissue.

The following examples are provided to exemplify various aspects of theinvention and are not intended to limit the scope of the invention.

EXAMPLES Example 1

To obtain human cDNA clones, a cDNA library was screened with a murineMDM2 (mMDM2) CDNA probe. A cDNA library was prepared by usingpolyadenylated RNA isolated from the human colonic carcinoma cell lineCaCo-2 as a template for the production of random hexamer primed doublestranded CDNA. Gubler and Hoffmann, 1983, Gene 25:263-268. The cDNA wasligated to adaptors and then to the lambda YES phage vector, packaged,and plated as described by Elledge et al. (Proc. Natl. Acad. Sci. USA,88:1731-1735, 1991). The library was screened initially with aP-labelled (Kinzler, K. W., et al., Nucl. Acids Res. 17:3645-3653(1989), Feinberg and Vogelstein, 1983, Anal. Biochem. 132:6-13) mMDM2cDNA probe (nucleotides 259 to 1508 (Fakharzadeh et al., 1991, EMBO J.10:1565-1569)) and then rescreened with an hMDM2 cDNA clone containingnucleotides 40 to 702.

Twelve clones were obtained, and one of the clones was used to obtainthirteen additional clones by re-screening the same library. In total,twenty-five clones were obtained, partially or totally sequenced, andmapped. Sequence analysis of the twenty-five clones revealed severalcDNA forms indicative of alternative splicing. The sequence shown inFIG. 1 is representative of the most abundant class and was assembledfrom three clones: c14-2 (nucleotides 1-949), c89 (nucleotides467-1737), and c33 (nucleotides 390-2372). The 3′ end of theuntranslated region has not yet been cloned in mouse or human. The 5′end is likely to be at or near nucleotide 1. There was an open readingframe extending from the 5″ end of the human CDNA sequence to nucleotide1784. Although the signal for translation initiation could not beunambiguously defined, the ATG at nucleotide 312 was considered the mostlikely position for several reasons. First, the sequence similaritybetween hMDM2 and mMDM2 fell off dramatically upstream of nucleotide312. This lack of conservation in an otherwise highly conserved proteinsuggested that the sequences upstream of the divergence may not code forprotein. Second, an anchored polymerase chain reaction (PCR) approachwas employed in an effort to acquire additional upstream cDNA sequence.Ochman et al., 1985, In: PCR Technology: Principles and Applications forDNA Amplification (Erlich, ed.) pp. 105-111 (Stockton, New York). The 5′ends of the PCR derived clones were very similar (within 3 bp) to the 5′ends of clones obtained from the cDNA library, suggesting that the 5′end of the hMDM2 sequence shown in FIG. 1 may represent the 5′ end ofthe transcript. Third, in vitro translation of the sequence shown inFIG. 1, beginning with the methionine encoded by the nucleotide 312 ATG,generated a protein similar in size to that observed in human cells.

In FIG. 1, hMDM2 cDNA sequence, hMDM2 and mMDM2 nucleotide and aminoacid sequences are compared. The mouse sequence is only shown where itdiffers from the corresponding human sequence. Asterisks mark the 5′ and3′ boundaries of the previously published mMDM2 cDNA. Fakharzadeh etal., 1991, EMBO J. 10:1565-1569. Dashes indicate insertions. The mouseand human amino acid sequences are compared from the putativetranslation start site at nucleotide 312 through the conserved stopcodon at nucleotide 1784.

Comparison of the human and mouse MDM2 coding regions revealedsignificant conservation at the nucleotide (80.3%) and amino acid(80.4%) levels. Although hMDM2 and mMDM2 bore little similarity to othergenes recorded in current databases, the two proteins shared severalmotifs. These included a basic nuclear location signal (Tanaka, 1990,FEBS Letters 271:41-46) at codons 181, to 185, several casein kinase IIserine phosphorylation sites (Pinna, 1990, Biochem. et. Biophys. Acta.1054:267-284) at codons 166 to 169, 192 to 195, 269 to 272, and 290 to293, an acidic activation domain (Ptashne, 1988, Nature 355:683-689) atcodons 223 to 274, and two metal binding sites (Harrison, 1991, Nature353:715) at codons 305 to 322 and 461 to 478, neither of which is highlyrelated to known DNA binding domains. The protein kinase A domain notedin mMDM2 (Fakharzadeh et al., 1991, EMBO J. 10:1565-1569) was notconserved in hMDM2.

Example 2

To determine whether the hMDM2 protein could bind to human p53 proteinin vitro, an hMDM2 expression vector was constructed from the cDNAclones. The hMDM2 expression vector was constructed in pBluescript SK+(Stratagene) from overlapping CDNA clones. The construct contained thesequence shown in FIG. 1 from nucleotide 312 to 2176. A 42 bp blackbettle virus ribosome entry sequence (Dasmahapatra et al., 1987, NucleicAcid Research 15:3933) was placed immediately upstream of this hMDM2sequence in order to obtain a high level of expression. This construct,as well as p53 (El-Deriy et al., 1992, Nature Genetics, in press) andMCC (Kinzler et al., 1991, Science 251:1366-1370) constructs inpBluescript SK+, were transcribed with T7 RNA polymerase and translatedin a rabbit reticulocyte lysate (Promega) according to themanufacturer's instructions.

Although the predicted size of the protein generated from the constructwas only 55.2 kd (extending from the methionine at nucleotide 312 tonucleotide 1784), in vitro translated protein migrated at approximately95 kilodaltons.

Ten μl of lysate containing the three proteins (hMDM2, p53 and MCC),alone or mixed in pairs, were incubated at 37° C. for 15 minutes. Onemicrogram (10 μL) of p53 Ab1 (monoclonal antibody specific for theC-terminus of p53) or Ab2 (monoclonal antibody specific for theN-terminus of p53) (Oncogene Science), or 5 μl of rabbit serumcontaining MDM2 Ab (polyclonal rabbit anti-hMDUM2 antibodies) orpreimmune rabbit serum (obtained from the rabbit which produced thehMDM2 Ab), were added as indicated. The polyclonal rabbit antibodieswere raised against an E. coli-produced hMDM2-glutathione S-transferasefusion protein containing nucleotides 390 to 816 of the hMDM2 cDNA.Ninety μl of RIPA buffer (10 mM tris [pH 7.5], 1% sodium deoxycholate,1% NP40, 150 mM NaCl, 0.1% SDS), SNNTE buffer, or Binding Buffer(El-Deriy et al., 1992, Nature Genetics, in press) were then added andthe mixtures allowed to incubate at 4° C. for 2 hours.

Two milligrams of protein A sepharose were added to each tube, and thetubes were rotated end-over-end at 4° C. for 1 hour. After pelleting andwashing, the immunoprecipitates were subjected to SDS-polyacrylamide gelelectrophoresis and the dried gels autoradiographed for 10 to 60 minutesin the presence of Enhance (New England Nuclear).

FIG. 2 shows the co-precipitation of hMDM2 and p53. The three buffersproduced similar results, although the co-precipitation was lessefficient in SNNTE buffer containing 0.5 M NaCl (FIG. 2, lanes 5 and 8)than in Binding Buffer containing 0.1 M NaCl (FIG. 2 lanes 6 and 9).

In vitro translated hMDM2, p53 and MCC proteins were mixed as indicatedabove and incubated with p53 Abl, p53 Ab2, hMDM2 Ab, or preimmune serum.Lanes 1, 4, 7, 10 and 14 contain aliquots of the protein mixtures usedfor immunoprecipitation. The bands running slightly faster than p53 arepolypeptides produced from internal translation initiation sites.

The hMDM2 protein was not immunoprecipitated with monoclonal antibodiesto either the C-terminal or N-terminal regions of p53 (FIG. 2, lanes 2and 3). However, when in vitro translated human p53 was mixed with thehMDM2 translation product, the anti-p53 antibodies precipitated hMDM2protein along with p53, demonstrating an association in vitro (FIG. 2,lanes 5 and 6). As a control, a protein of similar electrophoreticmobility from another gene (MCC (Kinzler et al., 1991, Science251:1366-1370)) was mixed with p53. No co-precipitation of the MCCprotein was observed (FIG. 2, lanes 8 and 9). When an in vitrotranslated mutant form of p53 (175^(his)) was mixed with hMDM2 protein,a similar co-precipitation of hMDM2 and p53 proteins was also observed.

In the converse of the experiments described above, the anti-hMDM2antibodies immunoprecipitated p53 when mixed with hMDM2 protein (FIG. 2,lane 15) but failed to precipitate p53 alone (FIG. 5, lane 13).Preimmune rabbit serum failed to precipitate either hMDM2 or p53 (FIG.2, lane 16).

Example 3

In order to ascertain the chromosomal localization of hMDM2, somaticcell hybrids were screened with an hMDM2 CDNA probe. A human-hamsterhybrid containing only human chromosome 12 was found to hybridize to theprobe. Screening of hybrids containing portions of chromosome 12(Turc-Carel et al., 1986, Cancer Genet. Cytogenet. 23:291-299) with thesame probe narrowed the localization to chromosome 12q12-14.

Example 4

Previous studies have shown that this region of chromosome 12 is oftenaberrant in human sarcomas. Mandahl et al., 1987, Genes Chromosomes &Cancer 1:9-14; Turc-Carel et al., 1986, Cancer Genet. Cytogenet.23:291-299; Meltzer et al., 1991, Cell Growth & Differentiation2:495-501. To evaluate the possibility that hMDM2 was geneticallyaltered in such cancers, Southern blot analysis was performed.

FIG. 3 shows examples of the amplification of the HMDM2 gene insarcomas. Cellular DNA (5 μg) was digested with EcoRI, separated byagarose gel electrophoresis, and transferred to nylon as described byReed and Mann (Nucl. Acids Res., 1985, 13:7207-7215). The cellular DNAwas derived from five primary sarcomas (lanes 1-4, 6) and one sarcomacell line (OsA-Cl, lane 5). The filters were then hybridized with anhMDM2 cDNA fragment probe nucleotide 1-949 (see FIG. 1), or to a controlprobe which identifies fragments of similar size (DCC gene, 1.65 CDNAfragment). Fearon, 1989, Science 247:49-56. Hybridization was performedas described by Vogelstein et al. (Cancer Research, 1987, 47:4806-4813).A striking amplification of hMDM2 sequences was observed in several ofthese tumors. (See FIG. 3, lanes 2, 3 and 5). Of 47 sarcomas analyzed,17 exhibited hMDM2 amplification ranging from 5 to 50 fold. These tumorsincluded 7 to 13 liposarcomas, 7 of 22 malignant fibrous histiocytomas(MFH), 3 of 11 osteosarcomas, and 0 and 1 rhabdomyosarcomas. Five benignsoft tissue tumors (ipomas) and twenty-seven carcinomas (colorectal orgastric) were also tested by Southern blot analysis and no amplificationwas observed.

Example 5

This example illustrates that gene amplification is associated withincreased expression.

FIG. 4A illustrates hMDM2 expression as demonstrated by Northern blotanalysis. Because of RNA degradation in the primary sarcomas, only thecell lines could be productively analyzed by Northern blot. RNA wasseparated by electrophoresis in a MOPS-formaldehyde gel andelectrophoretically transferred to nylon filters. Transfer andhybridization were performed as described by Kinzler et al. (Nature332:371-374, 1988). The RNA was hybridized to the hMDM2 fragmentdescribed in FIG. 3. Ten μg of total RNA derived, respectively, from twosarcoma cell lines (OsA-CL, lane 1 and RC13, lane 2) and the colorectalcancer cell line (CaCo-2) used to make the cDNA library (Lane 3). Lane 4contains 10 μg of polyadenylated CaCo-2 RNA. RNA sizes are shown in kb.In the one available sarcoma cell line with hMDM2 amplification, asingle transcript of approximately 5.5 kb was observed (FIG. 4A, lane1). The amount of this transcript was much higher than in a sarcoma cellline without amplification (FIG. 4A, lane 2) or in a carcinoma cell line(FIG. 4A, lane 3). When purified mRNA (rather than total RNA) from thecarcinoma cell line was used for analysis, an hMDM2 transcript of 5.5 kbcould also be observed (FIG. 4A, lane 4).

FIG. 4B illustrates hMDM2 expression as demonstrated by Western blotanalysis of the sarcoma cell lines RC13 (lane 1), OSA-CL (lane 3), HOS(lane 4), and the carcinoma cell line CaCo-2 (lane 2).

FIG. 4C illustrates hMDM2 expression as demonstrated by Western blotanalysis of primary sarcomas. Lanes 1 to 3 contain protein from sarcomaswith hMDM2 amplifications, and lanes 4 and 5 contain protein fromsarcomas without hMDM2 amplification.

Western blots using affinity purified MDM2 Ab were performed with 50 μgprotein per lane as described by Kinzler et al. (Mol. Cell. Biol., 1990,10:634-642), except that the membranes were blocked in 10% nonfat driedmilk and 10% goat serum, and secondary antibodies were coupled tohorseradish peroxidase, permitting chemiluminescent detection (AmershamECL). MDM2 Ab was affinity purified with a pATH-hMDM2 fusion proteinusing methods described in Kinzler et al. (Mol. Cell. Biol. 10:634-642,1990). Non-specifically reactive proteins of about 75-85, 105-120 and170-200 kd were observed in all lanes, irrespective of hMDM2amplification status. hMDM2 proteins, of about 90-97 kd, were observedonly in the hMDM2-amplified tumors. Protein marker sizes are shown inkd.

A protein of approximately 97 kilodaltons was expressed at high levelsin the sarcoma cell line with hMDM2 amplification (FIG. 4B, lane 3),whereas no expression was evident in two sarcoma cell lines withoutamplification or in the carcinoma cell line (FIG. 4B, lanes 1, 2 and 4).Five primary sarcomas were also examined by Western blot analysis. Threeprimary sarcomas with amplification expressed the same size protein asthat observed in the sarcoma cell line (FIG. 4C, lanes 1-3), while noprotein was observed in the two sarcomas without amplification (FIG. 4C,lanes 4 and 5).

Expression of the hMDM2 RNA in the sarcoma with amplification wasestimated to be at least 30 fold higher than that in the other linesexamined. This was consistent with the results of Western blot analysis.

The above examples demonstrate that hMDM2 binds to p53 in vitro and isgenetically altered (i.e., amplified) in a significant fraction ofsarcomas, including MFH, liposarcomas, and osteosarcomas. These are themost common sarcomas of soft tissue and bone. Weiss and Enzinger, 1978,Cancer 41:2250-2266; Malawer et al., 1985, In: Cancer: Principles andPractice of Oncology, DeVita et al., Eds., pp. 1293-1342 (Lippincott,Philadelphia).

Human MDM2 amplification is useful for understanding the pathogenesis ofthese often lethal cancers.

MDM2 may functionally inactivate p53 in ways similar to those employedby virally encoded oncoproteins such as SV40 T-antigen, adenovirus ElB,and HPV E6. Lane and Bechimol, 1990, Genes and Development 4:1-8;Werness et al., 1990, Science 248:76. Consistent with this hypothesis,no sarcomas with hMDM2 amplification had any of the p53 gene mutationsthat occur commonly in other tumors. hMDM2 amplification provides aparallel between viral carcinogenesis and the naturally occurringgenetic alterations underlying sporadic human cancer. The finding thatexpression of hMDM2 is correspondingly elevated in tumors withamplification of the gene are consistent with the finding that MDM2binds to p53, and with the hypothesis that overexpression of MDM2 insarcomas allows escape from p53 regulated growth control. This mechanismof tumorigenesis has striking parallels to that previously observed forvirally induced tumors (Lane and Bechimol, 1990, Genes and Development4:1-8; Werness et al., 1990, Science 248:76), in which viral oncogeneproducts bind to and functionally inactivate p53.

Example 6

This example demonstrates that MDM2 expression inhibits p53-mediatedtransactivation.

To determine if MDM2 could influence the ability of p53 to activatetranscription, expression vectors coding for the two proteins werestably transfected into yeast along with a p53-responsive reporterconstruct. The reporter consisted of a β-galactosidase gene under thetranscriptional control of a minimal promoter and a multimerized humanDNA sequence which strongly bound p53 in vitro (Kern, S. E., et al.,Science 256:827-830 (1992). Reporter expression was completely dependenton p53 in this assay (FIG. 5, compare bars a and c). MDM2 expression wasfound to inhibit p53-mediated transactivation of this reporter 16-foldrelative to isogeneic yeast lacking MDM2 expression (FIG. 5, comparebars a and b). Western blot analysis confirmed that p53 (53 kD) wasexpressed equivalently in strains with and without MDM2 (90 kD) (FIG. 1,inset).

METHODS. The MDM2 expression plasmid, pPGK-MDM2, was constructed byinserting the full length MDM2 cDNA (Oliner, J. D., et al., Nature358:80-83 (1992)) into pPGK (Poon, D. et al., Mol. and Cell. Biol.1111:4809-4821 (1991)), immediately downstream of the phosphoglyceratekinase constitutive promoter. Galactose-inducible p53 (pRS314SN, Nigro,J. M., et al., Mol. and Cell. Biol. 12:1357-1365 (1992)), lexA-VP16(YVLexA, Dalton, S., et al., Cell 68:597-612 (1992)), and lexA (YLexA,YVLexA minus VP16) plasmids were used as indicated. The reporters werePG16-lacZ (Kern, S. E. et al., Science 256:827-830 (1992))(p53-responsive) and pJK103 (Kamens, J., et al., Mol. Cell. Biol.10:2840-2847 (1990)) (lexA-responsive). S. cerevisiae strain pEGY48 wastransformed as described (Kinzler, K. W. et al., Nucl. Acids Res.17:3645-3653 (1989)). Yeast strains represented by bars a-c were grownat 30° C. to mid-log phase in selective liquid medium containing 2%raffinose as the carbon source, induced for 30 minutes by the additionof 2% galactose, harvested, and lysed as described (Kern, S. E. et al.,Science 256:827-830 (1992)). The strains represented by bars d-f weretreated similarly, except that the cells were induced in galactose for 4hours to obtain measurable levels of β-galactosidase. β-galactosidaseactivities shown represent the mean of three to five experimental values(error bars indicate s.e.m.). Protein concentrations were determined bya Coomassie blue-based (bio-Rad) assay. The β-galactosidase assays wereperformed with AMPGD chemiluminescent substrate and Emerald enhancerCropix) according to the manufacturer's instructions. β-galactosidaseactivities of bars b and c are shown relative to that of bar A;β-galactosidase activities of bars e and f are shown relative to that ofbar d. Western blots were performed as described (Oliner, J. D., et al.,Nature 358:80-83 (1992)), using p53 Ab1801 (lower panel, OncogeneScience) or MDM2 polyclonal antibodies (Oliner, J. D., et al., Nature358:80-83 (1992)) (upper panel).

To ensure that this inhibition was not simply a general transcriptionaldown regulation mediated by the expression of the foreign MDM2 gene, ayeast strain was created that contained a different transcriptionalactivator (lexA-VP16, consisting of the lexA DNA binding domain fused tothe VP16 acidic activation domain), a similar reporter (with alexA-responsive site upstream of a β-galactosidase gene), and the sameMDM2 expression vector. The results shown in FIG. 1 (bars d & e)demonstrate that lexA-VP16 transactivation was unaffected by thepresence of MDM2. Furthermore, MDM2 expression had no apparent effect onthe growth rate of the cells.

Example 7

This example demonstrates the domains of p53 and MDM2 which interactwith each other.

To gain insight into the mechanism of the MDM2-mediated p53 inhibition,the domains of MDM2 and p53 responsible for binding to one another weremapped. The yeast system used to detect protein-protein binding takesadvantage of the modular nature of transcription factor domains (Keegan,L., et al., Science 231:699-704 (1986); Chien, C.-T., Proc. Natl. Acad.Sci. U.S.A. 88:9578-9582 (1991); Brent, R., et al., Cell 43:729-731(1985); Ma, J., et al., Cell 55:4430446 (1988). Generically, if proteinI—(fused to a sequence-specific DNA binding domain) is capable ofbinding to protein 2 (fused to a transcriptional activation domain),then co-expression of both fusion proteins will result intranscriptional activation of a suitable reporter. In our experiments,the lexA DNA binding domain (amino acids 2-202) and the B42 acidicactivation domain (AAD) were used in the fusion constructs. The reporter(Kamens, I., et al., Mol. Cell. Biol. 10:2840-2847 (1990); contained alexA-responsive site upstream of a β-galactosidase gene. As an initialcontrol experiment, full length MDM2 was inserted into the lexA fusionvector, and full length p53, supplying its intrinsic activation domainwas inserted into a non-fusion vector. The combination resulted in theactivation of the lexA-responsive reporter, while the same expressionconstructs lacking either the MDM2 or p53 cDNA inserts failed toactivate β-galactosidase (Table I, stains 1, 2, and 3). Thus, activationwas dependent upon MDM2-p53 binding.

This assay was then applied to mapping the interaction domains of eachprotein. Full length CDNA fragments encoding MDM2 or p53 were randomlysheared by sonication, amplified by polymerase chain reaction, sizefractionated, cloned into the appropriate fusion vectors and transfectedinto yeast along with the reporter and the full length version of theother protein.

METHODS. Full length MDM2 CDNA in pBluescript SK+(Stratagene) wasdigested with XhoI and BamHI to excise the entire insert. After agarosegel purification, the insert was sheared into random fragments bysonication, polished with the Kilenow fragment of DNA polymerase I,ligated to catch linkers, and amplified by the polymerase chain reactionas described (Kinzler, K. W., et al., Nucl. Acids Res. 17:3645-3653(1989)). The fragments were fractionated on an acrylamide gel into sizeranges of 100-400 bp or 400-1000 pb, cloned into lexA(1-202)+PL (Ruden,D. M., et al., Nature 350:250-252 (1991)), and transfected into bacteria(XL-1 Blue, Stratagene). At least 10,000 bacterial colonies were scrapedoff agar plates, and the plasmid DNA was transfected into a strain ofpEGY48 containing pRS314N (p53 expression vector) and pJK103(lexA-responsive β-galactosidase reporter). Approximately 5,000 yeastclones were plated on selective medium containing 2% dextrose, and werereplica-plated onto glalctose- and X-gal-containing selective medium.Blue colonies (17) appeared only on the plates containing the largerfragments of MDM2. The 17 isolated colonies were tested for blue colorin this assay both in the presence and in the absence of galactose (p53induction); all tested positive in the presence of galactose but only 2of the 17 tested positive in its absence. MDM2-containing plasmid DNAextracted from the 17 yeast clones was selectively transferred tobacterial strain KC8 and sequenced from the lexA-MDM2 junction. TheeMDM2 sequences of the two p53-independent clones are diagrammed in FIG.6A. The MDM2 sequences of the remaining 15 p53-dependent clones codedfor peptides ranging from 135 to 265 a.a. in length and beganexclusively at the initiator methionine. Three of the MDM2 sequencesobtained are shown at the top of FIG. 6B. The lower 6 sequences weregenetically engineered (using the polymerase chain reaction andappropriate primers) into lexA(1-202)+PL and subsequently tested tofurther narrow the binding region.

Fragments of p53 were also cloned into pJG4-5, producing a fusionprotein C-terminal to the B42 acidic activation domain and incorporatingan epitope of hemagglutinin. The clones were transfected into a strainof pEGY48 already containing lex-MDM2 (plex-202+PL containing fulllength MDM2) and pJK103. The top three p53 sequences shown in FIG. 6C.were derived from yeast obtained by colony screening, whereas the lowerthree were genetically engineered to contain the indicated fragments.

The resultant yeast colonies were examined for β-galactosidase activityin situ. Of approximately 5000 clones containing MDM2 fragments fused tothe lexA DNA binding domain, 17 were found to score positively in thisassay. The clones could be placed into two classes. The first class (twoclones) expressed low levels of β-galactosidase (about 5-fold less thanthe other fifteen clones) and β-galactosidase expression was independentof p53 expression (FIG. 6A). These two clones encoded MDM2 amino acids190-340 and 269-379, respectively. The region shared between these twoclones overlapped the only acidic domain in MDM2 (amino acids 230-301).This domain consisted of 37.5% aspartic and glutamic acid residues butno basic amino acids. This acidic domain appears to activatetranscription only when isolated from the rest of the MDM2 sequence,because the entire MDM2 protein fused to lexA had no measurableβ-galactosidase activity in the same assay (Table I, strain 3). Theother class (15 clones) each contained the amino terminal region of MDM2(FIG. 6B). The β-galactosidase activity of these clones was dependent onp53 co-expression. To narrow down the region of interaction, wegenerated six additional clones by genetic engineering. The smallesttested region of MDM2 which could functionally interact with full lengthp53 contained MDM2 codons 1 to 118 (FIG. 6B). The relatively large sizeof the domain required for interaction was consistent with the fact thatwhen small sonicated fragments of MDM2 were used in the screening assay(200 bp instead of 600 bp average size), no positively scoring cloneswere obtained.

In a converse set of experiments, yeast clones containing fragments ofp53 fused to the B42 AAD were screened for lexA-responsive reporterexpression in the presence of a lexA-MDM2 fusion protein. Sequencing ofthe 14 clones obtained in the screen revealed that they could be dividedinto three subsets, one containing amino acids 1-41, a second containingamino acids 13-57, and a third containing amino acids 1-50 (FIG. 2C).The minimal overlap between these three fragments contained codons13-41. Although this minimal domain was apparently necessary forinteraction with MDM2, it was insufficient, as the fragments required9-12 amino acids on either side of codons 13-41 for activity (FIG. 6C).To further test the idea that the amino terminal region of p53 wasrequired for MDM2 binding, we generated an additional yeast strainexpressing the lexA-DNA binding domain fused to p53 codons 74-393) andthe B42 acidic activation domain fused to full length MDM2. Thesestrains failed to activate the same lexA-responsive reporter (Table 1,strain 8), as expected if the N-terminus of p53 were required for theinteraction.

TABLE I STRAIN p53 MDM2 NUMBER CONSTRUCT CONSTRUCT ACTIVATION 1 p53^(a)Vector^(b) − 2 p53^(a) lexA-MDM2^(b) + 3 Vector^(a) lexA-MDM2^(b) − 4p53^(a) lexA-MDM2 (1-118)^(b) + S Vector^(a) lexA-MDM2 (1-118)^(b) − 6B42-p53 (1-41)^(c) lexA-MDM2^(b) + 7 B42-p53 (1-41)^(c) Vector^(b) − 8lexA-p53 (74-393)^(b) B42-MDM2^(c) − 9 p53 (1-137)^(a) lexA-MDM2^(b) −The MDM2 and p53 proteins expressed in each strain, along with therelevant reporters, are indicated. Numbers in parentheses refer to theMDM2 or p53 amino acids encoded (absence of parentheses indicated fulllength protein, that is, MDM2 amino acids 1 to 491 or p53 amino acids 1to 393). The lexA-responsive β-galactosidase reporter plasmid (pJK103,Kamens, J., et al., Mol. Cell. Biol. 10:2840-2847 (1990)) was present inall strains. ^(a)pRS314 vector (Nigro, J.M., et al., Mol. and Cell.Biol. 12:1357-1365 (1992). ^(b)plex(1-202) + PL vector, containing lexADNA binding domain fused to insert (Ruden, D.M., et al., Nature350:250-252 (1991). ^(c)pJG4-5 vector, containing B42 activation domainfused to insert. ^(d)(+) indicates that colonies turned blue following24 hours of incubation on X-gal-containing selective medium, while (−)indicates that colonies remained white following 72 hours of incubation.

Sequence analysis showed that all p53 and MDM2 fragments noted in FIG. 6were ligated in frame and in the correct orientation relative to the B42and lexA domains, respectively. Additionally, all clones compared inFIG. 6 expressed the relevant proteins at similar levels, as shown byWestern blotting (FIG. 7).

The most striking results of these mapping experiments was that theregion of p53 required to bind MDM2 was almost identical to thepreviously identified acidic activation domain of p53 (amino acids 2042)(Unger, T., et al., EMBO J. 11:1383-1390 (1992); Miller, C. W., et al.,Proc. Am. Assoc. Cancer Res. 33:386 (1992). This suggested that MDM2inhibits p53-mediated transcriptional activation by concealing theactivation domain of p53 from the transcriptional machinery. If thiswere true, the p53 activation domain, in isolation from the rest of thep53 protein, should still be inhibitable by full length MDM2. To testthis hypothesis, we produced a hybrid protein containing the p53activation domain (codons 1-73) fused to the lexA-DNA binding domain.This construct exhibited strong transcriptional activation of alexA-responsive reporter (FIG. 8), as predicted from previousexperiments in which the p53 activation domain was fused to another DNAbinding domain (Fields, S., et al., Science 249:1046-1049 (1990);Raycroft, L., et al., Science 249:1049-1051 (1990)). The lexA-p53 DNAconstruct was stably expressed in yeast along with the full length MDM2expression vector (or the vector alone). MDM2 expression resulted in afive-fold decrease in reporter activity, demonstrating that MDM2 canspecifically inhibit the function of the p53 activation domainregardless of the adjacent protein sequences tethering p53 to DNA FIG.8).

METHODS. Strains were grown to mid-log phase in 2% dextrose beforeinduction of p53 expression for 2 hours by the addition of 2% galactose.The lex-p53 construct was identical to lex-VP16 (YVlexA, Dalton, S., etal., Cell 68:597-612 (1992)) except that VP16 sequences were replaced byp53 sequences encoding amino acids 1 to 73.

The results obtained in the experiments discussed herein raise aninteresting paradox. If MDM2 binds to (FIG. 6) and conceals (FIG. 8) thep53 activation domain from the transcriptional machinery, how could thelexA-MDM2-p53 complex activate transcription from the lexA-responsivereporter (Table I, strain 2)? Because the only functional activationdomain in the lexA-MDM2-p53 complex of strain 2 is expected to becontributed by p53, one might predict that it would be concealed bybinding to MDM2 and thereby fail to activate. A potential resolution ofthis paradox is afforded by knowledge that p53 exists as a homotetramer(Stenger, J. E., et al., Mol. Carcinogenesis 5:102-106 (1992);Sturzbecher, H. W. et al., Oncogene 7:1513-1523 (1992). Thus theactivation noted in the lexA-MDM2-p53 complex could be due to thepresence of four individual activation domains contributed by the p53tetramer, not all of which were concealed by MDM2. As a direct test ofthis issue, the domain of p53 required for homo-oligomerization(Stenger, J. E., et al., Mol. Carcinogenesis 5:102-106 (1992);Sturzbecher, H. W. et al., Oncogene 7:1513-1523 (1992) (the C-terminus)was removed from the p53 expression construct, so that it consisted ofonly codons 1-137. This truncated p53 polypeptide retained the entireactivation domain (as shown in FIG. 8, bar a) and the entire domainrequired for interaction with MDM2 (Table I, strain 6). Yet, whenallowed to interact with lexA-MDM2, no transactivation of thelexA-responsive reporter was observed (Table I, strain 9). Because p53did not inhibit lexA-MDM2 binding to the lexA reporter (Table 1, strain2), the result of strain 9 is likely to be due to a direct inhibition ofthe isolated p53 activation domain by MDM2.

Example 8

This example illustrates the production and characterization ofantibodies specific for MDM2 epitopes.

The antigen preparations used to intraperitoneally immunize female(BALB/c X C57BIJ6)F1 mice comprised bacterially expressed,glutathione-column purified glutathione-S-transferase-MDM2 (GST-MDM2)fusion protein. (One preparation was further purified on apolyacrylamide gel and electroeluted.) The fusion protein contains a 16kD amino terminal portion of human MDM2 protein (amino acids 27 to 168).For immunization, the fusion protein was mixed with Ribi adjuvant (RibiImmunochem Research, Inc.).

Two mice were sacrificed and their spleen cells fused to SP2/0s myelomacells (McKenzie, et al., Oncogene, 4:543-548, 1989). Resultinghybridomas were screened by ELISA on trpE-MDM2 fusion protein-coatedmicrotiter wells. The trpE-MDM2 fusion protein contains the same portionof MDM2 as the GST-MDM2 fusion protein. Antigen was coated at aconcentration of 1 μg/ml.

A second fusion was performed as described except hybridomas werescreened on electroeluted, glutathione purified GST-MDM2. Positivehybridomas from both fusions were expanded and single cell subcloned.Subclones were tested by Western Blot for specificity to the 55 kDtrpE-MDM2 and the 43 kD GST-MDM2 fusion proteins.

Two Western Blot positive subclones (IF2 and JG3) were put into mice forascites generation. The resulting ascites were protein A purified. Bothpurified monoclonal antibodies tested positive by Western Blot andimmunoprecipitation for the 90 kD migrating MDM2 protein present in ahuman osteosarcoma cell line (OsA-CL), which overexpresses MDM2, andnegative in the HOS osteosarcoma, which does not overexpress MDM2.

ED9 was protein G-purified from ascites and found to be specific incryostat immunohistochemistry for MDM2 in osteosarcoma cells, as wasIF2.

Example 9

This example demonstrates the expression and detection of MDM2 at thecellular level.

To evaluate MDM2 expression at the cellular level, we producedmonoclonal antibodies against bacterially generated fusion proteinscontaining residues 27 to 168 of MDM2. (See example 8.) Of severalantibodies tested, mAb IF-2 was the most useful, as it detected MDM2 inseveral assays. For initial testing, we compared proteins derived fromOsA-CL, a sarcoma cell line with MDM2 amplification but without p53mutation (Table II) and proteins from SW480, a colorectal cancer cellline with p53 mutation (Barak et al., EMBO 12:461-468 (1993)) butwithout MDM2 amplification (data not shown). We could not distinguishwhether the low molecular weight bands in OsA-CL were due to proteindegradation or alternative processing of MDM2 transcripts. The more than20-fold difference in intensity between the signals observed in OsA-CLand SW480 is consistent with the greater than 20-fold difference in MDM2gene copy number in these two lines. Conversely, the 53 kd signaldetected with p53-specific mAb 1801 was much stronger in SW480 than inOsA-CL consistent with the presence of a mutated p53 in SW480.

Cells grown on cover slips were then used to assess the cellularlocalization of the MDM2 protein. A strong signal, exclusively nuclear,was observed in OsA-CL cells with the IF-2 mAb and a weaker signal,again strictly nuclear, was observed in SW480. The nuclear localizationof MDM2 is consistent with previous studies of mouse cells (Barak etal., EMBO 12:461-468 (1993)) and the fact that human MDM2 contains anuclear localization signal at residues 179 to 186. Reactivity with thep53-specific antibody was also confined to the nuclei of these two celllines, with the relative intensities consistent with the Western blotresults.

The IF-2 mAb was then used (at 5 μg/ml) to stain the seven primarysarcomas noted above. The nuclei of two of them (tumors #3 and #10)stained strongly. Both of these tumors contained MDM2 gene amplification(Table II). In the five tumors without amplification, little or no MDM2reactivity was observed.

TABLE II TUMOR TUMOR MDM2 P53 OVER- # ID TYPE^(a) AMPLIFICATION^(b)ALTERATION^(c) EXPRESSION^(d) 1 M-2 MFH ABSENT DELETION/ NONEREARRANGEMENT 2 M-5 MFH ABSENT CGC-CUC MUTATION; p53 Arg(158)-His 3 M-7MFH PRESENT NONE OBSERVED MDM2 4 M-8 MFH ABSENT DELETION NONE 5 M-14 MFWABSENT NONE OBSERVED N.T. 6 M-15 MFH ABSENT DELETION N.T. 7 M-16 MFHABSENT NONE OBSERVED NONE 8 M-17 MFH ABSENT NONE OBSERVED N.T. 9 M-18MFH ABSENT OVEREXPRESSED p53 10 M-20 MFH PRESENT NONE OBSERVED MDM2 11L-5 LIPOSARCOMA ABSENT NONE OBSERVED N.T. 12 L-7 LIPOSARCOMA ABSENTAAC-AGC MUTATION; N.T. Asn(239)-Ser. 13 L-9 LIPOSARCOMA PRESENT NONEOBSERVED N.T. 14 L-11 LIPOSARCOMA ABSENT NONE OBSERVED N.T. 15 KL5BLIPOSARCOMA ABSENT CAG-UAG MUTATION; N.T. Gln(144)-Stop 16 KL7LIPOSARCOMA PRESENT NONE OBSERVED N.T. 17 KL11 LIPOSARCOMA ABSENT NONEOBSERVED N.T. 18 KL11 LIPOSARCOMA ABSENT GGT-GAT MUTATION; EXON 5 N.T.SPLICE DONOR SITE 19 KL12 LIPOSARCOMA ABSENT NONE OBSERVED N.T. 20 KL28LIPOSARCOMA PRESENT NONE OBSERVED N.T. 21 KL30 LIPOSARCOMA PRESENT NONEOBSERVED 22 S189 LIPQSARCOMA PRESENT NONE OBSERVED N.T. 23 S131BLIPOSARCOMA ABSENT NONE OBSERVED N.T. 24 OSA-CL MFH PRESENT NONEOBSERVED MDM2 ^(a) MFH = malignant fibrous histiocytoma ^(b)as assessedby Southern blot ^(c)as assessed by Southern blot, sequencing of exons5-8, or immunohistochemical analysis ^(d)as assessed byimmunohistocheiaical analysis; N.T. = not tested

5 64 amino acids amino acid single linear protein NO NO N-terminal Homosapiens 17q 1 Met Glu Glu Pro Gln Ser Asp Pro Ser Val Glu Pro Pro LeuSer Gln 1 5 10 15 Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu AsnAsn Val Leu 20 25 30 Ser Pro Leu Pro Ser Gln Ala Met Asp Asp Leu Met LeuSer Pro Asp 35 40 45 Asp Ile Glu Gln Trp Phe Thr Glu Asp Pro Gly Pro AspGlu Ala Pro 50 55 60 2372 base pairs nucleic acid double linear cDNA NONO Homo sapiens CaCo-2 12q12-14 CDS 312..1784 2 GCACCGCGCG AGCTTGGCTGCTTCTGGGGC CTGTGTGGCC CTGTGTGTCG GAAAGATGGA 60 GCAAGAAGCC GAGCCCGAGGGGCGGCCGCG ACCCCTCTGA CCGAGATCCT GCTGCTTTCG 120 CAGCCAGGAG CACCGTCCCTCCCCGGATTA GTGCGTACGA GCGCCCAGTG CCCTGGCCCG 180 GAGAGTGGAA TGATCCCCGAGGCCCAGGGC GTCGTGCTTC CGCAGTAGTC AGTCCCCGTG 240 AAGGAAACTG GGGAGTCTTGAGGGACCCCC GACTCCAAGC GCGAAAACCC CGGATGGTGA 300 GGAGCAGGCA A ATG TGC AATACC AAC ATG TCT GTA CCT ACT GAT GGT GCT 350 Met Cys Asn Thr Asn Met SerVal Pro Thr Asp Gly Ala 1 5 10 GTA ACC ACC TCA CAG ATT CCA GCT TCG GAACAA GAG ACC CTG GTT AGA 398 Val Thr Thr Ser Gln Ile Pro Ala Ser Glu GlnGlu Thr Leu Val Arg 15 20 25 CCA AAG CCA TTG CTT TTG AAG TTA TTA AAG TCTGTT GGT GCA CAA AAA 446 Pro Lys Pro Leu Leu Leu Lys Leu Leu Lys Ser ValGly Ala Gln Lys 30 35 40 45 GAC ACT TAT ACT ATG AAA GAG GTT CTT TTT TATCTT GGC CAG TAT ATT 494 Asp Thr Tyr Thr Met Lys Glu Val Leu Phe Tyr LeuGly Gln Tyr Ile 50 55 60 ATG ACT AAA CGA TTA TAT GAT GAG AAG CAA CAA CATATT GTA TAT TGT 542 Met Thr Lys Arg Leu Tyr Asp Glu Lys Gln Gln His IleVal Tyr Cys 65 70 75 TCA AAT GAT CTT CTA GGA GAT TTG TTT GGC GTG CCA AGCTTC TCT GTG 590 Ser Asn Asp Leu Leu Gly Asp Leu Phe Gly Val Pro Ser PheSer Val 80 85 90 AAA GAG CAC AGG AAA ATA TAT ACC ATG ATC TAC AGG AAC TTGGTA GTA 638 Lys Glu His Arg Lys Ile Tyr Thr Met Ile Tyr Arg Asn Leu ValVal 95 100 105 GTC AAT CAG CAG GAA TCA TCG GAC TCA GGT ACA TCT GTG AGTGAG AAC 686 Val Asn Gln Gln Glu Ser Ser Asp Ser Gly Thr Ser Val Ser GluAsn 110 115 120 125 AGG TGT CAC CTT GAA GGT GGG AGT GAT CAA AAG GAC CTTGTA CAA GAG 734 Arg Cys His Leu Glu Gly Gly Ser Asp Gln Lys Asp Leu ValGln Glu 130 135 140 CTT CAG GAA GAG AAA CCT TCA TCT TCA CAT TTG GTT TCTAGA CCA TCT 782 Leu Gln Glu Glu Lys Pro Ser Ser Ser His Leu Val Ser ArgPro Ser 145 150 155 ACC TCA TCT AGA AGG AGA GCA ATT AGT GAG ACA GAA GAAAAT TCA GAT 830 Thr Ser Ser Arg Arg Arg Ala Ile Ser Glu Thr Glu Glu AsnSer Asp 160 165 170 GAA TTA TCT GGT GAA CGA CAA AGA AAA CGC CAC AAA TCTGAT AGT ATT 878 Glu Leu Ser Gly Glu Arg Gln Arg Lys Arg His Lys Ser AspSer Ile 175 180 185 TCC CTT TCC TTT GAT GAA AGC CTG GCT CTG TGT GTA ATAAGG GAG ATA 926 Ser Leu Ser Phe Asp Glu Ser Leu Ala Leu Cys Val Ile ArgGlu Ile 190 195 200 205 TGT TGT GAA AGA AGC AGT AGC AGT GAA TCT ACA GGGACG CCA TCG AAT 974 Cys Cys Glu Arg Ser Ser Ser Ser Glu Ser Thr Gly ThrPro Ser Asn 210 215 220 CCG GAT CTT GAT GCT GGT GTA AGT GAA CAT TCA GGTGAT TGG TTG GAT 1022 Pro Asp Leu Asp Ala Gly Val Ser Glu His Ser Gly AspTrp Leu Asp 225 230 235 CAG GAT TCA GTT TCA GAT CAG TTT AGT GTA GAA TTTGAA GTT GAA TCT 1070 Gln Asp Ser Val Ser Asp Gln Phe Ser Val Glu Phe GluVal Glu Ser 240 245 250 CTC GAC TCA GAA GAT TAT AGC CTT AGT GAA GAA GGACAA GAA CTC TCA 1118 Leu Asp Ser Glu Asp Tyr Ser Leu Ser Glu Glu Gly GlnGlu Leu Ser 255 260 265 GAT GAA GAT GAT GAG GTA TAT CAA GTT ACT GTG TATCAG GCA GGG GAG 1166 Asp Glu Asp Asp Glu Val Tyr Gln Val Thr Val Tyr GlnAla Gly Glu 270 275 280 285 AGT GAT ACA GAT TCA TTT GAA GAA GAT CCT GAAATT TCC TTA GCT GAC 1214 Ser Asp Thr Asp Ser Phe Glu Glu Asp Pro Glu IleSer Leu Ala Asp 290 295 300 TAT TGG AAA TGC ACT TCA TGC AAT GAA ATG AATCCC CCC CTT CCA TCA 1262 Tyr Trp Lys Cys Thr Ser Cys Asn Glu Met Asn ProPro Leu Pro Ser 305 310 315 CAT TGC AAC AGA TGT TGG GCC CTT CGT GAG AATTGG CTT CCT GAA GAT 1310 His Cys Asn Arg Cys Trp Ala Leu Arg Glu Asn TrpLeu Pro Glu Asp 320 325 330 AAA GGG AAA GAT AAA GGG GAA ATC TCT GAG AAAGCC AAA CTG GAA AAC 1358 Lys Gly Lys Asp Lys Gly Glu Ile Ser Glu Lys AlaLys Leu Glu Asn 335 340 345 TCA ACA CAA GCT GAA GAG GGC TTT GAT GTT CCTGAT TGT AAA AAA ACT 1406 Ser Thr Gln Ala Glu Glu Gly Phe Asp Val Pro AspCys Lys Lys Thr 350 355 360 365 ATA GTG AAT GAT TCC AGA GAG TCA TGT GTTGAG GAA AAT GAT GAT AAA 1454 Ile Val Asn Asp Ser Arg Glu Ser Cys Val GluGlu Asn Asp Asp Lys 370 375 380 ATT ACA CAA GCT TCA CAA TCA CAA GAA AGTGAA GAC TAT TCT CAG CCA 1502 Ile Thr Gln Ala Ser Gln Ser Gln Glu Ser GluAsp Tyr Ser Gln Pro 385 390 395 TCA ACT TCT AGT AGC ATT ATT TAT AGC AGCCAA GAA GAT GTG AAA GAG 1550 Ser Thr Ser Ser Ser Ile Ile Tyr Ser Ser GlnGlu Asp Val Lys Glu 400 405 410 TTT GAA AGG GAA GAA ACC CAA GAC AAA GAAGAG AGT GTG GAA TCT AGT 1598 Phe Glu Arg Glu Glu Thr Gln Asp Lys Glu GluSer Val Glu Ser Ser 415 420 425 TTG CCC CTT AAT GCC ATT GAA CCT TGT GTGATT TGT CAA GGT CGA CCT 1646 Leu Pro Leu Asn Ala Ile Glu Pro Cys Val IleCys Gln Gly Arg Pro 430 435 440 445 AAA AAT GGT TGC ATT GTC CAT GGC AAAACA GGA CAT CTT ATG GCC TGC 1694 Lys Asn Gly Cys Ile Val His Gly Lys ThrGly His Leu Met Ala Cys 450 455 460 TTT ACA TGT GCA AAG AAG CTA AAG AAAAGG AAT AAG CCC TGC CCA GTA 1742 Phe Thr Cys Ala Lys Lys Leu Lys Lys ArgAsn Lys Pro Cys Pro Val 465 470 475 TGT AGA CAA CCA ATT CAA ATG ATT GTGCTA ACT TAT TTC CCC 1784 Cys Arg Gln Pro Ile Gln Met Ile Val Leu Thr TyrPhe Pro 480 485 490 TAGTTGACCT GTCTATAAGA GAATTATATA TTTCTAACTATATAACCCTA GGAATTTAGA 1844 CAACCTGAAA TTTATTCACA TATATCAAAG TGAGAAAATGCCTCAATTCA CATAGATTTC 1904 TTCTCTTTAG TATAATTGAC CTACTTTGGT AGTGGAATAGTGAATACTTA CTATAATTTG 1964 ACTTGAATAT GTAGCTCATC CTTTACACCA ACTCCTAATTTTAAATAATT TCTACTCTGT 2024 CTTAAATGAG AAGTACTTGG TTTTTTTTTT CTTAAATATGTATATGACAT TTAAATGTAA 2084 CTTATTATTT TTTTTGAGAC CGAGTCTTGC TCTGTTACCCAGGCTGGAGT GCAGTGGGTG 2144 ATCTTGGCTC ACTGCAAGCT CTGCCCTCCC CGGGTTCGCACCATTCTCCT GCCTCAGCCT 2204 CCCAATTAGC TTGGCCTACA GTCATCTGCC ACCACACCTGGCTAATTTTT TGTACTTTTA 2264 GTAGAGACAG GGTTTCACCG TGTTAGCCAG GATGGTCTCGATCTCCTGAC CTCGTGATCC 2324 GCCCACCTCG GCCTCCCAAA GTGCTGGGAT TACAGGCATGAGCCACCG 2372 491 amino acids amino acid linear protein 3 Met Cys AsnThr Asn Met Ser Val Pro Thr Asp Gly Ala Val Thr Thr 1 5 10 15 Ser GlnIle Pro Ala Ser Glu Gln Glu Thr Leu Val Arg Pro Lys Pro 20 25 30 Leu LeuLeu Lys Leu Leu Lys Ser Val Gly Ala Gln Lys Asp Thr Tyr 35 40 45 Thr MetLys Glu Val Leu Phe Tyr Leu Gly Gln Tyr Ile Met Thr Lys 50 55 60 Arg LeuTyr Asp Glu Lys Gln Gln His Ile Val Tyr Cys Ser Asn Asp 65 70 75 80 LeuLeu Gly Asp Leu Phe Gly Val Pro Ser Phe Ser Val Lys Glu His 85 90 95 ArgLys Ile Tyr Thr Met Ile Tyr Arg Asn Leu Val Val Val Asn Gln 100 105 110Gln Glu Ser Ser Asp Ser Gly Thr Ser Val Ser Glu Asn Arg Cys His 115 120125 Leu Glu Gly Gly Ser Asp Gln Lys Asp Leu Val Gln Glu Leu Gln Glu 130135 140 Glu Lys Pro Ser Ser Ser His Leu Val Ser Arg Pro Ser Thr Ser Ser145 150 155 160 Arg Arg Arg Ala Ile Ser Glu Thr Glu Glu Asn Ser Asp GluLeu Ser 165 170 175 Gly Glu Arg Gln Arg Lys Arg His Lys Ser Asp Ser IleSer Leu Ser 180 185 190 Phe Asp Glu Ser Leu Ala Leu Cys Val Ile Arg GluIle Cys Cys Glu 195 200 205 Arg Ser Ser Ser Ser Glu Ser Thr Gly Thr ProSer Asn Pro Asp Leu 210 215 220 Asp Ala Gly Val Ser Glu His Ser Gly AspTrp Leu Asp Gln Asp Ser 225 230 235 240 Val Ser Asp Gln Phe Ser Val GluPhe Glu Val Glu Ser Leu Asp Ser 245 250 255 Glu Asp Tyr Ser Leu Ser GluGlu Gly Gln Glu Leu Ser Asp Glu Asp 260 265 270 Asp Glu Val Tyr Gln ValThr Val Tyr Gln Ala Gly Glu Ser Asp Thr 275 280 285 Asp Ser Phe Glu GluAsp Pro Glu Ile Ser Leu Ala Asp Tyr Trp Lys 290 295 300 Cys Thr Ser CysAsn Glu Met Asn Pro Pro Leu Pro Ser His Cys Asn 305 310 315 320 Arg CysTrp Ala Leu Arg Glu Asn Trp Leu Pro Glu Asp Lys Gly Lys 325 330 335 AspLys Gly Glu Ile Ser Glu Lys Ala Lys Leu Glu Asn Ser Thr Gln 340 345 350Ala Glu Glu Gly Phe Asp Val Pro Asp Cys Lys Lys Thr Ile Val Asn 355 360365 Asp Ser Arg Glu Ser Cys Val Glu Glu Asn Asp Asp Lys Ile Thr Gln 370375 380 Ala Ser Gln Ser Gln Glu Ser Glu Asp Tyr Ser Gln Pro Ser Thr Ser385 390 395 400 Ser Ser Ile Ile Tyr Ser Ser Gln Glu Asp Val Lys Glu PheGlu Arg 405 410 415 Glu Glu Thr Gln Asp Lys Glu Glu Ser Val Glu Ser SerLeu Pro Leu 420 425 430 Asn Ala Ile Glu Pro Cys Val Ile Cys Gln Gly ArgPro Lys Asn Gly 435 440 445 Cys Ile Val His Gly Lys Thr Gly His Leu MetAla Cys Phe Thr Cys 450 455 460 Ala Lys Lys Leu Lys Lys Arg Asn Lys ProCys Pro Val Cys Arg Gln 465 470 475 480 Pro Ile Gln Met Ile Val Leu ThrTyr Phe Pro 485 490 1710 base pairs nucleic acid double linear cDNA NONO Mus musculus CDS 202..1668 4 GAGGAGCCGC CGCCTTCTCG TCGCTCGAGCTCTGGACGAC CATGGTCGCT CAGGCCCCGT 60 CCGCGGGGCC TCCGCGCTCC CCGTGAAGGGTCGGAAGATG CGCGGGAAGT AGCAGCCGTC 120 TGCTGGGCGA GCGGGAGACC GACCGGACACCCCTGGGGGA CCCTCTCGGA TCACCGCGCT 180 TCTCCTGCGG CCTCCAGGCC A ATG TGC AATACC AAC ATG TCT GTG TCT ACC 231 Met Cys Asn Thr Asn Met Ser Val Ser Thr1 5 10 GAG GGT GCT GCA AGC ACC TCA CAG ATT CCA GCT TCG GAA CAA GAG ACT279 Glu Gly Ala Ala Ser Thr Ser Gln Ile Pro Ala Ser Glu Gln Glu Thr 1520 25 CTG GTT AGA CCA AAA CCA TTG CTT TTG AAG TTG TTA AAG TCC GTT GGA327 Leu Val Arg Pro Lys Pro Leu Leu Leu Lys Leu Leu Lys Ser Val Gly 3035 40 GCG CAA AAC GAC ACT TAC ACT ATG AAA GAG ATT ATA TTT TAT ATT GGC375 Ala Gln Asn Asp Thr Tyr Thr Met Lys Glu Ile Ile Phe Tyr Ile Gly 4550 55 CAG TAT ATT ATG ACT AAG AGG TTA TAT GAC GAG AAG CAG CAG CAC ATT423 Gln Tyr Ile Met Thr Lys Arg Leu Tyr Asp Glu Lys Gln Gln His Ile 6065 70 GTG TAT TGT TCA AAT GAT CTC CTA GGA GAT GTG TTT GGA GTC CCG AGT471 Val Tyr Cys Ser Asn Asp Leu Leu Gly Asp Val Phe Gly Val Pro Ser 7580 85 90 TTC TCT GTG AAG GAG CAC AGG AAA ATA TAT GCA ATG ATC TAC AGA AAT519 Phe Ser Val Lys Glu His Arg Lys Ile Tyr Ala Met Ile Tyr Arg Asn 95100 105 TTA GTG GCT GTA AGT CAG CAA GAC TCT GGC ACA TCG CTG AGT GAG AGC567 Leu Val Ala Val Ser Gln Gln Asp Ser Gly Thr Ser Leu Ser Glu Ser 110115 120 AGA CGT CAG CCT GAA GGT GGG AGT GAT CTG AAG GAT CCT TTG CAA GCG615 Arg Arg Gln Pro Glu Gly Gly Ser Asp Leu Lys Asp Pro Leu Gln Ala 125130 135 CCA CCA GAA GAG AAA CCT TCA TCT TCT GAT TTA ATT TCT AGA CTG TCT663 Pro Pro Glu Glu Lys Pro Ser Ser Ser Asp Leu Ile Ser Arg Leu Ser 140145 150 ACC TCA TCT AGA AGG AGA TCC ATT AGT GAG ACA GAA GAG AAC ACA GAT711 Thr Ser Ser Arg Arg Arg Ser Ile Ser Glu Thr Glu Glu Asn Thr Asp 155160 165 170 GAG CTA CCT GGG GAG CGG CAC CGG AAG CGC CGC AGG TCC CTG TCCTTT 759 Glu Leu Pro Gly Glu Arg His Arg Lys Arg Arg Arg Ser Leu Ser Phe175 180 185 GAT CCG AGC CTG GGT CTG TGT GAG CTG AGG GAG ATG TGC AGC GGCGGC 807 Asp Pro Ser Leu Gly Leu Cys Glu Leu Arg Glu Met Cys Ser Gly Gly190 195 200 ACG AGC AGC AGT AGC AGC AGC AGC AGC GAG TCC ACA GAG ACG CCCTCG 855 Thr Ser Ser Ser Ser Ser Ser Ser Ser Glu Ser Thr Glu Thr Pro Ser205 210 215 CAT CAG GAT CTT GAC GAT GGC GTA AGT GAG CAT TCT GGT GAT TGCCTG 903 His Gln Asp Leu Asp Asp Gly Val Ser Glu His Ser Gly Asp Cys Leu220 225 230 GAT CAG GAT TCA GTT TCT GAT CAG TTT AGC GTG GAA TTT GAA GTTGAG 951 Asp Gln Asp Ser Val Ser Asp Gln Phe Ser Val Glu Phe Glu Val Glu235 240 245 250 TCT CTG GAC TCG GAA GAT TAC AGC CTG AGT GAC GAA GGG CACGAG CTC 999 Ser Leu Asp Ser Glu Asp Tyr Ser Leu Ser Asp Glu Gly His GluLeu 255 260 265 TCA GAT GAG GAT GAT GAG GTC TAT CGG GTC ACA GTC TAT CAGACA GGA 1047 Ser Asp Glu Asp Asp Glu Val Tyr Arg Val Thr Val Tyr Gln ThrGly 270 275 280 GAA AGC GAT ACA GAC TCT TTT GAA GGA GAT CCT GAG ATT TCCTTA GCT 1095 Glu Ser Asp Thr Asp Ser Phe Glu Gly Asp Pro Glu Ile Ser LeuAla 285 290 295 GAC TAT TGG AAG TGT ACC TCA TGC AAT GAA ATG AAT CCT CCCCTT CCA 1143 Asp Tyr Trp Lys Cys Thr Ser Cys Asn Glu Met Asn Pro Pro LeuPro 300 305 310 TCA CAC TGC AAA AGA TGC TGG ACC CTT CGT GAG AAC TGG CTTCCA GAC 1191 Ser His Cys Lys Arg Cys Trp Thr Leu Arg Glu Asn Trp Leu ProAsp 315 320 325 330 GAT AAG GGG AAA GAT AAA GTG GAA ATC TCT GAA AAA GCCAAA CTG GAA 1239 Asp Lys Gly Lys Asp Lys Val Glu Ile Ser Glu Lys Ala LysLeu Glu 335 340 345 AAC TCA GCT CAG GCA GAA GAA GGC TTG GAT GTG CCT GATGGC AAA AAG 1287 Asn Ser Ala Gln Ala Glu Glu Gly Leu Asp Val Pro Asp GlyLys Lys 350 355 360 CTG ACA GAG AAT GAT GCT AAA GAG CCA TGT GCT GAG GAGGAC AGC GAG 1335 Leu Thr Glu Asn Asp Ala Lys Glu Pro Cys Ala Glu Glu AspSer Glu 365 370 375 GAG AAG GCC GAA CAG ACG CCC CTG TCC CAG GAG AGT GACGAC TAT TCC 1383 Glu Lys Ala Glu Gln Thr Pro Leu Ser Gln Glu Ser Asp AspTyr Ser 380 385 390 CAA CCA TCG ACT TCC AGC AGC ATT GTT TAT AGC AGC CAAGAA AGC GTG 1431 Gln Pro Ser Thr Ser Ser Ser Ile Val Tyr Ser Ser Gln GluSer Val 395 400 405 410 AAA GAG TTG AAG GAG GAA ACG CAG CAC AAA GAC GAGAGT GTG GAA TCT 1479 Lys Glu Leu Lys Glu Glu Thr Gln His Lys Asp Glu SerVal Glu Ser 415 420 425 AGC TTC TCC CTG AAT GCC ATC GAA CCA TGT GTG ATCTGC CAG GGG CGG 1527 Ser Phe Ser Leu Asn Ala Ile Glu Pro Cys Val Ile CysGln Gly Arg 430 435 440 CCT AAA AAT GGC TGC ATT GTT CAC GGC AAG ACT GGACAC CTC ATG TCA 1575 Pro Lys Asn Gly Cys Ile Val His Gly Lys Thr Gly HisLeu Met Ser 445 450 455 TGT TTC ACG TGT GCA AAG AAG CTA AAA AAA AGA AACAAG CCC TGC CCA 1623 Cys Phe Thr Cys Ala Lys Lys Leu Lys Lys Arg Asn LysPro Cys Pro 460 465 470 GTG TGC AGA CAG CCA ATC CAA ATG ATT GTG CTA AGTTAC TTC AAC 1668 Val Cys Arg Gln Pro Ile Gln Met Ile Val Leu Ser Tyr PheAsn 475 480 485 TAGCTGACCT GCTCACAAAA ATAGAATTTT ATATTTCTAA CT 1710 489amino acids amino acid linear protein 5 Met Cys Asn Thr Asn Met Ser ValSer Thr Glu Gly Ala Ala Ser Thr 1 5 10 15 Ser Gln Ile Pro Ala Ser GluGln Glu Thr Leu Val Arg Pro Lys Pro 20 25 30 Leu Leu Leu Lys Leu Leu LysSer Val Gly Ala Gln Asn Asp Thr Tyr 35 40 45 Thr Met Lys Glu Ile Ile PheTyr Ile Gly Gln Tyr Ile Met Thr Lys 50 55 60 Arg Leu Tyr Asp Glu Lys GlnGln His Ile Val Tyr Cys Ser Asn Asp 65 70 75 80 Leu Leu Gly Asp Val PheGly Val Pro Ser Phe Ser Val Lys Glu His 85 90 95 Arg Lys Ile Tyr Ala MetIle Tyr Arg Asn Leu Val Ala Val Ser Gln 100 105 110 Gln Asp Ser Gly ThrSer Leu Ser Glu Ser Arg Arg Gln Pro Glu Gly 115 120 125 Gly Ser Asp LeuLys Asp Pro Leu Gln Ala Pro Pro Glu Glu Lys Pro 130 135 140 Ser Ser SerAsp Leu Ile Ser Arg Leu Ser Thr Ser Ser Arg Arg Arg 145 150 155 160 SerIle Ser Glu Thr Glu Glu Asn Thr Asp Glu Leu Pro Gly Glu Arg 165 170 175His Arg Lys Arg Arg Arg Ser Leu Ser Phe Asp Pro Ser Leu Gly Leu 180 185190 Cys Glu Leu Arg Glu Met Cys Ser Gly Gly Thr Ser Ser Ser Ser Ser 195200 205 Ser Ser Ser Glu Ser Thr Glu Thr Pro Ser His Gln Asp Leu Asp Asp210 215 220 Gly Val Ser Glu His Ser Gly Asp Cys Leu Asp Gln Asp Ser ValSer 225 230 235 240 Asp Gln Phe Ser Val Glu Phe Glu Val Glu Ser Leu AspSer Glu Asp 245 250 255 Tyr Ser Leu Ser Asp Glu Gly His Glu Leu Ser AspGlu Asp Asp Glu 260 265 270 Val Tyr Arg Val Thr Val Tyr Gln Thr Gly GluSer Asp Thr Asp Ser 275 280 285 Phe Glu Gly Asp Pro Glu Ile Ser Leu AlaAsp Tyr Trp Lys Cys Thr 290 295 300 Ser Cys Asn Glu Met Asn Pro Pro LeuPro Ser His Cys Lys Arg Cys 305 310 315 320 Trp Thr Leu Arg Glu Asn TrpLeu Pro Asp Asp Lys Gly Lys Asp Lys 325 330 335 Val Glu Ile Ser Glu LysAla Lys Leu Glu Asn Ser Ala Gln Ala Glu 340 345 350 Glu Gly Leu Asp ValPro Asp Gly Lys Lys Leu Thr Glu Asn Asp Ala 355 360 365 Lys Glu Pro CysAla Glu Glu Asp Ser Glu Glu Lys Ala Glu Gln Thr 370 375 380 Pro Leu SerGln Glu Ser Asp Asp Tyr Ser Gln Pro Ser Thr Ser Ser 385 390 395 400 SerIle Val Tyr Ser Ser Gln Glu Ser Val Lys Glu Leu Lys Glu Glu 405 410 415Thr Gln His Lys Asp Glu Ser Val Glu Ser Ser Phe Ser Leu Asn Ala 420 425430 Ile Glu Pro Cys Val Ile Cys Gln Gly Arg Pro Lys Asn Gly Cys Ile 435440 445 Val His Gly Lys Thr Gly His Leu Met Ser Cys Phe Thr Cys Ala Lys450 455 460 Lys Leu Lys Lys Arg Asn Lys Pro Cys Pro Val Cys Arg Gln ProIle 465 470 475 480 Gln Met Ile Val Leu Ser Tyr Phe Asn 485

What is claimed is:
 1. A nucleic acid molecule comprising nucleotides 1to 2372 as shown in SEQ ID NO:2 or a fragment thereof comprising atleast 34 contiguous nucleotides of SEQ ID NO:2.
 2. The nucleic acidmolecule of claim 1 which comprises the coding sequence of human MDM2.3. The nucleic acid molecule of claim 1 which is complementary to thecoding sequence of human MDM2.
 4. The nucleic acid molecule of claim 1which comprises at least 34 contiguous nucleotides which arecomplementary to the coding sequence of human MDM2.
 5. The nucleic acidmolecule of claim 1 which comprises at least 40 contiguous nucleotideswhich are complementary to the coding sequence of human MDM2.
 6. Thenucleic acid molecule of claim 1 which comprises at least 50 contiguousnucleotides which are complementary to the coding sequence of humanMDM2.
 7. The nucleic acid molecule of claim 1 which comprises at least100 contiguous nucleotides which are complementary to the codingsequence of human MDM2.
 8. A nucleic acid molecule consisting of atleast 14 contiguous nucleotides of the sequence of human MDM2, as shownin SEQ ID NO:2.
 9. An antisense oligonucleotide which binds to a humanMDM2 mRNA and prevents translation of the human MDM2 MRNA.
 10. Theantisense oligonucleotide of claim 9 which consists of at least 14contiguous nucleotides of the sequence of human MDM2 as shown in SEQ IDNO:2.
 11. The nucleic acid molecule of claim 2 which is a ribo-nucleicacid molecule.